Crotamine as a vehicle for non-viral gene delivery for Pompe disease

Genetic deficiency of lysosomal acid alpha glucosidase or acid maltase (GAA) results in Pompe disease (PD), encompassing at least five clinical subtypes of varying severity. The current approved enzyme replacement therapy (ERT) for PD is via IV infusion every 2 weeks of a recombinant human GAA (rhGAA) secreted by Chinese hamster ovary (CHO) cells (alglucosidase alfa/Myozyme, Sanofi/Genzyme). Although alglucosidase alfa has proven to be efficient in rescuing cardiac abnormalities and extending the life span of the infantile form, the response in skeletal muscle is variable. ERT usually begins when the patients are symptomatic and secondary problems are already present which are compounded by low alglucosidase alfa uptake, transient nature (every 2 weeks with a rapid return to defect levels), variable glycogen reduction, autophagic accumulation, immune response and high cost. A consensus at a recent US Acid Maltase Deficiency (AMD) conference suggested that a multi-pronged approach including gene therapy, diet, exercise, etc. must be evaluated for a successful treatment of PD. Compared to replication defective viruses, non-viral gene transfer offers fewer safety concerns and, if recent studies are validated, has a wider range of cells. In order for gene therapy (GT) to succeed, the gene of interest must be delivered into the affected cell and expressed to overcome the inherited deficiency. Cell penetrating peptides (CPPs) enter eukaryotic cells through an energy-independent mechanism and efficiently carry biologically active and therapeutic molecules into cells and localize in the cytoplasm or nucleus. CPPs are usually covalently linked to the cargo, including peptides and DNA. Crotamine (Cro) from the South American rattlesnake-Crotalus durrissus terrificus venom, can bind electrostatically to plasmid DNA to deliver into cells, including muscle. We have assembled a bacterial expression vector for Cro and purified the recombinant Cro (rCro). Transient transfection in AMD fibroblasts and ex vivo in whole blood from an adult Pompe patient with rCro complexed with the pcDNA3 x hGAA cDNA demonstrated increased GAA activity. In GAA knockout (KO) mice receiving a single injection of rCro complexed to pcDNA3 x hGAA cDNA intraperitoneally (IP), we found increased GAA activity in tissues after 48 hr. After 8 treatments-IP over 55 days, we found increased vertical hang-time activity, reduced glycogen deposition, increased GAA activity/hGAA plasmid in tissues and minimal immune-reaction to rCro. A subsequent study of 5 administrations every 2 to 3 weeks showed reverse of the clinical phenotypes by running wheel activity, Rotarod, grip-strength meter, open field mobility and T-maze. Tissue culture experiments in PD fibroblast, lymphoid and skeletal muscle cell lines showed increased GAA activity after rCro transient gene delivery.

A rare case of infantile onset Pompe disease with genetic diagnosis

Glycogen storage disease type II, also called Pompe disease or acid maltase deficiency is a disorder of muscle glycogenoses with a wide range of clinical manifestations. It is one of the disorders of glycogen metabolism caused by a deficiency of lysosomal acid α-1, 4-glucosidase (acid maltase) resulting in lysosomal glycogen accumulation in cardiac, skeletal and smooth muscle cells. The pattern of inheritance is autosomal recessive with a gene for enzyme located on chromosome 17q25.2.It is the first recognized lysosomal storage disorder and the first neuromuscular disorder for which enzyme replacement therapy has been approved. We report a case of four month old female child, born to primi gravida third degree consanguineous couple, who presented with history of respiratory illness, hypotonia and developmental delay. Baby was sick needing mechanical ventilation and inotropic support. Echocardiography showed concentric LV hypertrophy with no LV outflow tract obstruction. In view of consanguinity, developmental delay, hepatomegaly and cardiomegaly, provisional diagnosis of a storage disorder, probably infantile Pompe disease was considered. Dried blood spot for α-1, 4-glucosidase enzyme assay confirmed the same. Enzyme replacement therapy was considered, but child progressed to cardiac failure needing prolonged ventilation and expired on day 8 of admission. Whole genome exome sequencing revealed 2 mutations which confirmed the diagnosis. Infantile Pompe disease is fatal without treatment. High index of suspicion and early diagnosis may help in taking advantage of emerging therapeutics, such as ERT which is capable of changing the natural history of the disease.

Identification of patients with Pompé disease using routine pathology results: PATHFINDER (creatine kinase) study

AimsAdult-onset inherited errors of metabolism can be difficult to diagnose. Some cases of potentially treatable myopathy are caused by autosomal recessive acid α-1,4 glucosidase (acid maltase) deficiency (Pompé disease). This study investigated whether screening of asymptomatic patients with elevated creatine kinase (CK) could improve detection of Pompé disease.MethodsPathology databases in six hospitals were used to identify patients with elevated CK results (>2× upper limit of normal). Patients were recalled for measurement of acid α-1,4 glucosidase activity in dried blood spot samples.ResultsSamples were obtained from 812 patients with elevated CK. Low α-glucosidase activity was found in 13 patients (1.6%). Patients with neutropaenia (n=4) or who declined further testing (n=1) were excluded. Confirmation plasma specimens were obtained from eight individuals (1%) for a white cell lysosomal enzyme panel, and three (0.4%) were confirmed to have low α-1,4-glucosidase activity. One patient was identified as a heterozygous carrier of an acid α-1,4 glucosidase c.-32–13 G>T mutation. Screening also identified one patient who was found to have undiagnosed Fabry disease and one patient with McArdle’s disease. One patient later presented with Pompé’s after an acute illness. Including the latent case, the frequency of cases at 0.12% was lower than the 2.5% found in studies of patients with raised CK from neurology clinics (p<0.001).ConclusionsScreening pathology databases for elevated CK may identify patients with inherited metabolic errors affecting muscle metabolism. However, the frequency of Pompé’s disease identified from laboratory populations was less than that in patients referred for neurological investigation.

Late-onset Pompe disease: preliminary results of enzyme replacement therapy

Pompe disease is an orphan hereditary accumulation disease associated with a deficiency of the lysosomal enzyme alglucosidase alpha. Manifestations of the disease are associated with pathological deposition of glycogen in body tissues as a result of GAA gene mutation and subsequent reduction in the activity of the enzyme alglucosidase alpha or acid maltase. The variety of phenotypic forms and varying degrees of damage to the skeletal and respiratory muscles, cardiomyocytes and internal organs greatly complicates the diagnosis and treatment of patients with Pompe»s disease. This article describes the clinical case of late-onset Pompe disease, which was followed by a course of enzyme replacement therapy, as well as an assessment of the condition before and after treatment and preliminary results.

Dyspnea

The most common causes of dyspnea are not neuromuscular, but rather cardiac and pulmonary. However, dyspnea is an important and serious manifestation of many neuromuscular disorders, and it may compound an underlying pulmonary or cardiac problem. The diaphragm is a skeletal muscle under the control of a peripheral nerve and may be targeted by inflammatory neuropathies such as Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), and brachial plexitis or myopathies such as acid maltase deficiency, muscular dystrophy (MD), and neuromuscular disorders such as myasthenia gravis (MG). Periodic measurement of pulmonary function is a recommended measure in neuromuscular clinics.

Reevaluating Muscle Biopsies in the Diagnosis of Pompe Disease: A Corroborative Report

Background: Previous reports suggest that although a diagnostic muscle biopsy can confirm the presence of Pompe disease, the absence of a definitive biopsy result does not rule out the diagnosis. Methods: In this study, we reviewed patients with a limb-girdle syndrome who demonstrated nonspecific abnormalities of muscle, without evidence of the classical changes of acid maltase deficiency. These patients were rescreened for Pompe disease using dried blood spot (DBS) testing. Results: Twenty-seven patients provided blood samples for the DBS test. Four patients underwent subsequent genetic testing. Genetic analysis demonstrated that one patient tested positive for Pompe disease and one patient had one copy of a pathogenic variant. Conclusions: In conclusion, the ability of a diagnostic muscle biopsy to definitively rule out the presence of Pompe disease is limited. There is a role for a screening DBS in all patients presenting with a limb-girdle syndrome without a clear diagnosis.